Chemical arrays and methods of using the same

ABSTRACT

Methods and compositions for generating mixtures of product molecules from an initial chemical array are provided. In the subject methods, a chemical array of surface immobilized first moieties is subjected to cleavage conditions such that a composition of solution phase first moieties is produced. The resultant composition of solution phase first moieties is then contacted with one or more reactants to produce a mixture of product molecules that are different from the first moieties. Also provided are the arrays employed in the subject methods and kits for practicing the subject methods.

INTRODUCTION

Chemical arrays, such as nucleic acid and protein arrays, are findingincreasing use in a variety of different applications, and in doing soare making a significant impact in a variety of different fields,including research, medicine, and the like. In many instances, arraysinclude regions of usually different composition arranged in apredetermined configuration on a substrate. These regions (sometimesreferenced as “features”) are positioned at known respective locations(“addresses”) on the substrate and are therefore “addressable.”

In using such arrays, the arrays are, in many applications, exposed to asample. Upon sample exposure, the arrays will exhibit an observedbinding pattern that is dependent on the sample composition to whichthey have been contacted. This observed binding pattern is then detectedupon interrogating the array. In the example of a nucleic acid array,the observed binding pattern is then employed to determine the presenceand/or concentration of one or more polynucleotide components of thesample. Representative methods for sample preparation, labeling, andhybridizing include those disclosed in U.S. Pat. Nos. 6,201,112;6,132,997; and 6,235,483; as well as published United States patentapplication 20020192650.

Arrays can be fabricated by depositing previously obtained biopolymersonto a substrate, or by in situ synthesis methods. The in situfabrication methods include those described in U.S. Pat. Nos. 5,449,754and 6,180,351 as well as published PCT application no. WO 98/41531 andthe references cited therein. Further details of fabricating biopolymerarrays are described in U.S. Pat. Nos. 6,242,266; 6,232,072; 6,180,351and U.S. Pat. No. 6,171,797. Other techniques for fabricating biopolymerarrays include known light directed synthesis techniques.

As the technology of making and using arrays continues to advance, thereis a continued interest in the development of new applications for thesepowerful tools.

SUMMARY OF THE INVENTION

Methods and compositions for generating mixtures of product moleculesfrom an initial chemical array are provided. In the subject methods, achemical array of surface immobilized first moieties is subjected tocleavage conditions such that a composition of solution phase firstmoieties is produced. The resultant composition of solution phase firstmoieties is then contacted with one or more reactants to produce amixture of product molecules that are different from the first moieties.Also provided are the arrays employed in the subject methods and kitsfor practicing the subject methods.

Aspects of the invention include: (a) subjecting an array of a pluralityof features each including first moieties immobilized on a surface of asolid support via a cleavable domain having a cleavable region toconditions sufficient to cleave the cleavable linker and generate asolution phase composition of said first moieties; and (b) contactingthe resultant solution phase composition of first moieties with one ormore reactants to produce a mixture of product molecules that aredifferent from the first moieties. In certain embodiments, the firstmoieties are polymers, e.g., biopolymers, such as nucleic acids orpolypeptides. In certain embodiments, the cleavable region of thecleavable domain is chemically cleavable, e.g., it is a base or acidlabile linker, while in other embodiments it is enzymatically cleavable.In certain embodiments, the one or more reactants comprises an enzymaticactivity, as is found in an enzymatic reaction system, such as atemplate dependent polymerase reaction system. In certain embodiments,the product molecules are nucleic acids, such as deoxyribonucleic acidsor ribonucleic acids.

Also provided are arrays that include a plurality of nucleic acidfeatures each including nucleic acids immobilized on a surface ofsubstrate via a cleavable linker. In representative embodiments, thesurface immobilized single-stranded nucleic acids are described by theformula:surface-L-V-wherein:

L is a cleavable domain having a cleavable region; and

V is a variable domain;

where the single-stranded nucleic acid may be oriented with its 3′ or 5′end proximal to the substrate surface and the variable domain V of thesurface immobilized single-stranded nucleic acids differs betweenfeatures.

Also provided are kits that include: (a) an as described above; and (b)a cleavage agent for cleaving the cleavable linker. In certainembodiments, the kits further include one or more members of anenzymatic reaction system, as reviewed above.

DEFINITIONS

A “biopolymer” is a polymer of one or more types of repeating units.Biopolymers are typically found in biological systems and particularlyinclude polysaccharides (such as carbohydrates), and peptides (whichterm is used to include polypeptides, and proteins whether or notattached to a polysaccharide) and polynucleotides as well as theiranalogs such as those compounds composed of or containing amino acidanalogs or non-amino acid groups, or nucleotide analogs ornon-nucleotide groups. As such, this term includes polynucleotides inwhich the conventional backbone has been replaced with a non-naturallyoccurring or synthetic backbone, and nucleic acids (or synthetic ornaturally occurring analogs) in which one or more of the conventionalbases has been replaced with a group (natural or synthetic) capable ofparticipating in Watson-Crick type hydrogen bonding interactions.Polynucleotides include single or multiple stranded configurations,where one or more of the strands may or may not be completely alignedwith another. Specifically, a “biopolymer” includes DNA (includingcDNA), RNA and oligonucleotides, regardless of the source.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymercomposed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean apolymer composed of deoxyribonucleotides.

The term “mRNA” means messenger RNA.

A “biomonomer” references a single unit, which can be linked with thesame or other biomonomers to form a biopolymer (for example, a singleamino acid or nucleotide with two linking groups one or both of whichmay have removable protecting groups). A biomonomer fluid or biopolymerfluid reference a liquid containing either a biomonomer or biopolymer,respectively (typically in solution).

A “nucleotide” refers to a sub-unit of a nucleic acid and has aphosphate group, a 5 carbon sugar and a nitrogen containing base, aswell as functional analogs (whether synthetic or naturally occurring) ofsuch sub-units which in the polymer form (as a polynucleotide) canhybridize with naturally occurring polynucleotides in a sequencespecific manner analogous to that of two naturally occurringpolynucleotides.

An “oligonucleotide” generally refers to a nucleotide multimer of about10 to 100 nucleotides in length, while a “polynucleotide” includes anucleotide multimer having any number of nucleotides.

A chemical “array”, unless a contrary intention appears, includes anyone, two or three-dimensional arrangement of addressable regions bearinga particular chemical moiety or moieties (for example, biopolymers suchas polynucleotide sequences) associated with that region. For example,each region may extend into a third dimension in the case where thesubstrate is porous while not having any substantial third dimensionmeasurement (thickness) in the case where the substrate is non-porous.An array is “addressable” in that it has multiple regions (sometimesreferenced as “features” or “spots” of the array) of different moieties(for example, different polynucleotide sequences) such that a region ata particular predetermined location (an “address”) on the array willdetect a particular target or class of targets (although a feature mayincidentally detect non-targets of that feature). The target for whicheach feature is specific is, in representative embodiments, known. Anarray feature is generally homogenous in composition and concentrationand the features may be separated by intervening spaces (although arrayswithout such separation can be fabricated).

In the case of an array, the “target” will be referenced as a moiety ina mobile phase (typically fluid), to be detected by probes (“targetprobes”) which are bound to the substrate at the various regions.However, either of the “target” or “target probes” may be the one whichis to be detected by the other (thus, either one could be an unknownmixture of polynucleotides to be detected by binding with the other).“Addressable set of probes” and analogous terms refers to the multipleregions of different moieties supported by or intended to be supportedby the array surface.

An “array layout” or “array characteristics”, refers to one or morephysical, chemical or biological characteristics of the array, such aspositioning of some or all the features within the array and on asubstrate, one or more feature dimensions, or some indication of anidentity or function (for example, chemical or biological) of a moietyat a given location, or how the array should be handled (for example,conditions under which the array is exposed to a sample, or arrayreading specifications or controls following sample exposure).

“Hybridizing” and “binding”, with respect to polynucleotides, are usedinterchangeably.

A “plastic” is any synthetic organic polymer of high molecular weight(for example at least 1,000 grams/mole, or even at least 10,000 or100,000 grams/mole.

“Flexible” with reference to a substrate or substrate web (including ahousing or one or more housing component such as a housing base and/orcover), references that the substrate can be bent 180 degrees around aroller of less than 1.25 cm in radius. The substrate can be so bent andstraightened repeatedly in either direction at least 100 times withoutfailure (for example, cracking) or plastic deformation. This bendingmust be within the elastic limits of the material. The foregoing testfor flexibility is performed at a temperature of 20° C. “Rigid” refersto a substrate (including a housing or one or more housing componentsuch as a housing base and/or cover) which is not flexible, and isconstructed such that a segment about 2.5 by 7.5 cm retains its shapeand cannot be bent along any direction more than 60 degrees (and oftennot more than 40, 20, 10, or 5 degrees) without breaking.

When one item is indicated as being “remote” from another, thisdescriptor indicates that the two items are at least in differentbuildings, and may be at least one mile, ten miles, or at least onehundred miles apart. When different items are indicated as being “local”to each other they are not remote from one another (for example, theycan be in the same building or the same room of a building).“Communicating”, “transmitting” and the like, of information referenceconveying data representing information as electrical or optical signalsover a suitable communication channel (for example, a private or publicnetwork, wired, optical fiber, wireless radio or satellite, orotherwise). Any communication or transmission can be between deviceswhich are local or remote from one another. “Forwarding” an item refersto any means of getting that item from one location to the next, whetherby physically transporting that item or using other known methods (wherethat is possible) and includes, at least in the case of data, physicallytransporting a medium carrying the data or communicating the data over acommunication channel (including electrical, optical, or wireless).“Receiving” something means it is obtained by any possible means, suchas delivery of a physical item (for example, an array or array carryingpackage). When information is received it may be obtained as data as aresult of a transmission (such as by electrical or optical signals overany communication channel of a type mentioned herein), or it may beobtained as electrical or optical signals from reading some other medium(such as a magnetic, optical, or solid state storage device) carryingthe information. However, when information is received from acommunication it is received as a result of a transmission of thatinformation from elsewhere (local or remote).

When two items are “associated” with one another they are provided insuch a way that it is apparent one is related to the other such as whereone references the other. For example, an array identifier can beassociated with an array by being on the array assembly (such as on thesubstrate or a housing) that carries the array or on or in a package orkit carrying the array assembly. Items of data are “linked” to oneanother in a memory when a same data input (for example, filename ordirectory name or search term) retrieves those items (in a same file ornot) or an input of one or more of the linked items retrieves one ormore of the others. In particular, when an array layout is “linked” withan identifier for that array, then an input of the identifier into aprocessor which accesses a memory carrying the linked array layoutretrieves the array layout for that array.

A “computer”, “processor” or “processing unit” are used interchangeablyand each references any hardware or hardware/software combination whichcan control components as required to execute recited steps. For examplea computer, processor, or processor unit includes a general purposedigital microprocessor suitably programmed to perform all of the stepsrequired of it, or any hardware or hardware/software combination whichwill perform those or equivalent steps. Programming may be accomplished,for example, from a computer readable medium carrying necessary programcode (such as a portable storage medium) or by communication from aremote location (such as through a communication channel).

A “memory” or “memory unit” refers to any device which can storeinformation for retrieval as signals by a processor, and may includemagnetic or optical devices (such as a hard disk, floppy disk, CD, orDVD), or solid state memory devices (such as volatile or non-volatileRAM). A memory or memory unit may have more than one physical memorydevice of the same or different types (for example, a memory may havemultiple memory devices such as multiple hard drives or multiple solidstate memory devices or some combination of hard drives and solid statememory devices).

An array “assembly” includes a substrate and at least one chemical arrayon a surface thereof. Array assemblies may include one or more chemicalarrays present on a surface of a device that includes a pedestalsupporting a plurality of prongs, e.g., one or more chemical arrayspresent on a surface of one or more prongs of such a device. An assemblymay include other features (such as a housing with a chamber from whichthe substrate sections can be removed). “Array unit” may be usedinterchangeably with “array assembly”.

“Reading” signal data from an array refers to the detection of thesignal data (such as by a detector) from the array. This data may besaved in a memory (whether for relatively short or longer terms).

A “package” is one or more items (such as an array assembly optionallywith other items) all held together (such as by a common wrapping orprotective cover or binding). Normally the common wrapping will also bea protective cover (such as a common wrapping or box) which will provideadditional protection to items contained in the package from exposure tothe external environment. In the case of just a single array assembly apackage may be that array assembly with some protective covering overthe array assembly (which protective cover may or may not be anadditional part of the array unit itself).

It will also be appreciated that throughout the present application,that words such as “cover”, “base” “front”, “back”, “top”, “upper”, and“lower” are used in a relative sense only.

“May” refers to optionally.

When two or more items (for example, elements or processes) arereferenced by an alternative “or”, this indicates that either could bepresent separately or any combination of them could be present togetherexcept where the presence of one necessarily excludes the other orothers.

The term “stringent assay conditions” as used herein refers toconditions that are compatible to produce binding pairs of nucleicacids, e.g., surface bound and solution phase nucleic acids, ofsufficient complementarity to provide for the desired level ofspecificity in the assay while being less compatible to the formation ofbinding pairs between binding members of insufficient complementarity toprovide for the desired specificity. Stringent assay conditions are thesummation or combination (totality) of both hybridization and washconditions.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization (e.g., as inarray, Southern or Northern hybridizations) are sequence dependent, andare different under different experimental parameters. Stringenthybridization conditions that can be used to identify nucleic acidswithin the scope of the invention can include, e.g., hybridization in abuffer comprising 50% formamide, 5×SSC, and 1% SDS at 42° C., orhybridization in a buffer comprising 5×SSC and 1% SDS at 65° C., bothwith a wash of 0.2×SSC and 0.1% SDS at 65° C. Exemplary stringenthybridization conditions can also include a hybridization in a buffer of40% formamide, 1 M NaCl, and 1% SDS at 37° C., and a wash in 1×SSC at45° C. Alternatively, hybridization to filter-bound DNA in 0.5 M NaHPO4,7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in0.1×SSC/0.1% SDS at 68° C. can be employed. Yet additional stringenthybridization conditions include hybridization at 60° C. or higher and3×SSC (450 mM sodium chloride/45 mM sodium citrate) or incubation at 42°C. in a solution containing 30% formamide, 1M NaCl, 0.5% sodiumsarcosine, 50 mM MES, pH 6.5. Those of ordinary skill will readilyrecognize that alternative but comparable hybridization and washconditions can be utilized to provide conditions of similar stringency.

In certain embodiments, the stringency of the wash conditions sets forththe conditions that determine whether a nucleic acid is specificallyhybridized to a surface bound nucleic acid. Wash conditions used toidentify nucleic acids may include, e.g.: a salt concentration of about0.02 molar at pH 7 and a temperature of at least about 50° C. or about55° C. to about 60° C.; or, a salt concentration of about 0.15 M NaCl at72° C. for about 15 minutes; or, a salt concentration of about 0.2′SSCat a temperature of at least about 50° C. or about 55° C. to about 60°C. for about 15 to about 20 minutes; or, the hybridization complex iswashed twice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. Stringent conditions for washing can also be,e.g., 0.2×SSC/0.1% SDS at 42° C.

A specific example of stringent assay conditions is rotatinghybridization at 65° C. in a salt based hybridization buffer with atotal monovalent cation concentration of 1.5 M (e.g., as described inU.S. patent application Ser. No. 09/655,482 filed on Sep. 5, 2000, thedisclosure of which is herein incorporated by reference) followed bywashes of 0.5×SSC and 0.1×SSC at room temperature.

Stringent assay conditions are hybridization conditions that are atleast as stringent as the above representative conditions, where a givenset of conditions are considered to be at least as stringent ifsubstantially no additional binding complexes that lack sufficientcomplementarity to provide for the desired specificity are produced inthe given set of conditions as compared to the above specificconditions, where by “substantially no more” is meant less than about5-fold more, typically less than about 3-fold more. Other stringenthybridization conditions are known in the art and may also be employed,as appropriate.

Stringent hybridization conditions are hybridization conditions that areat least as stringent as the above representative conditions, whereconditions are considered to be at least as stringent if they are atleast about 80% as stringent, typically at least about 90% as stringentas the above specific stringent conditions. Other stringenthybridization conditions are known in the art and may also be employed,as appropriate.

As such, the term “hybridization” refers to the formation of a duplexstructure by two single stranded nucleic acids due to complementary basepairing. Hybridization can occur between exactly complementary nucleicacid strands or between nucleic acid strands that contain minor regionsof mismatch. As used herein, the term “substantially complementary”refers to sequences that are complementary except for minor regions ofmismatch, wherein the total number of mismatched nucleotides is no morethan about 3 for a sequence about 15 to about 35 nucleotides in length.Conditions under which only exactly complementary nucleic acid strandswill hybridize are referred to as “stringent” or “sequence-specific”hybridization conditions. Stable duplexes of substantially complementarynucleic acids can be achieved under less stringent hybridizationconditions. Those skilled in the art of nucleic acid technology candetermine duplex stability empirically considering a number of variablesincluding, for example, the length and base pair concentration of theoligonucleotides, ionic strength, and incidence of mismatched basepairs. Computer software for calculating duplex stability iscommercially available from a variety of vendors.

Stringent, sequence-specific hybridization conditions, under which anoligonucleotide will hybridize only to the exactly complementary targetsequence, are well known in the art (see, e.g., Sambrook et al., 2001,Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., incorporated herein by reference). Stringentconditions are sequence dependent and will be different in differentcircumstances. Generally, stringent conditions are selected to be about5° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the base pairshave dissociated. Relaxing the stringency of the hybridizing conditionsallows sequence mismatches to be tolerated; the degree of mismatchtolerated can be controlled by suitable adjustment of the hybridizationconditions.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions for generating mixtures of product moleculesfrom an initial chemical array are provided. In the subject methods, achemical array of surface immobilized first moieties is subjected tocleavage conditions such that a composition of solution phase firstmoieties is produced. The resultant composition of solution phase firstmoieties is then contacted with one or more reactants to produce amixture of product molecules that are different from the first moieties.Also provided are the arrays employed in the subject methods and kitsfor practicing the subject methods.

Before the present invention is further described, it is to beunderstood that this invention is not limited to the particularembodiments described herein, as such may, of course, vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular representative embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible. The figuresshown herein are not necessarily drawn to scale, with some componentsand features being exaggerated for clarity.

As summarized above, the subject invention provides array-based methodsfor generating or producing pluralities of distinct product moleculesfrom an initial population of first moieties. By plurality is meant atleast 2, such as at least 5, including at least 10, where the number ofdistinct product molecules in the plurality may be at least about 25, atleast about 50, at least about 100, at least about 500, at least about1000 or more, such as at least about 5,000, at least about 10,000, atleast about 25,000 or more.

The subject methods of producing the above-described pluralities arearray-based methods, where a feature of the subject methods is that achemical array is employed as a source of first moieties from which theproduct molecules are produced via a chemical reaction that employs onor more reactants, as described in greater detail below. The termchemical array is, as described above, a composition of matter thatincludes a plurality of distinct feature of chemical moieties, referredto herein conveniently as first moieties. In representative embodiments,the chemical moieties are biopolymers, e.g., polypeptides or nucleicacids. In representative embodiments, the nucleic acids are, asdescribed above, ribonucleic acids or deoxyribonucleic acids. Theparticular nature of the chemical moieties of the array employed in thesubject methods necessarily depends on the nature of the productcomposition to be produced. For example, when the product composition isa nucleic acid composition, the chemical array employed is, in manyembodiments, a nucleic acid array. Alternatively, when the productcomposition is a polypeptide composition, the chemical array employedis, in many embodiments, a polypeptide array.

In practicing the subject methods, the first step of representativeembodiments is to subject a chemical array of first moieties to cleavageconditions sufficient to cleave or separate the surface immobilizedfirst moieties of the features of the array from the solid support toproduce a product composition of solution phase first moieties, e.g., byaction of a restriction endonuclease, by action of a chemical cleavageagent, as elaborated further below. The resultant composition ofsolution phase first moieties is then contacted with one or morereactants in a reaction step to produce a composition of productmolecules. Each of these steps of this representative embodiment is nowdescribed separately in greater detail.

The initial array employed in the first step of the subject methods,which may conveniently be referred to as the solution phase first moietygeneration step, is (in representative embodiments) a substrate having aplanar surface on which is immobilized a plurality of distinct chemicalfeatures of surface immobilized first moieties. As indicated above, thesurface immobilized first moieties may be a variety of different typesof compounds, but in representative embodiments are biopolymericcompounds, such as polypeptides and nucleic acids. Accordingly, for easeof convenience in further describing the invention, the invention willbe described in terms of the representative nucleic acid embodiments.However, in certain embodiments, the biopolymeric compounds are notnucleic acids, but instead non-nucleic acid biopolymers, e.g.,polypeptides.

The surface immobilized nucleic acids of a given feature on the arrayare made up of single-stranded nucleic acids, and in representativeembodiments single stranded deoxyribonucleic acids (where asingle-stranded nucleic acid is a nucleic acid that is not hybridized toa second, non-covalently bound nucleic acid). The surface immobilizedsingle-stranded nucleic acids are characterized by including: (a) avariable domain; and (b) a cleavable domain, where the cleavable domainincludes a region (e.g., site or sequence) that is cleavable, e.g., suchthat the cleavable domain serves as a cleavable linker; where thevariable domain is separated from the array surface by the cleavabledomain. The cleavable domain may or may not be a constant domain, asdesired. As such, in certain embodiments, the cleavable domain will bethe same or identical for all of the surface-immobilized compounds ofthe array, while in other embodiments the various surface displayedcompounds, or sets thereof, may have differing cleavable domains.

The initial arrays employed in the subject methods may be generated denovo or obtained as a pre-made array from a commercial source, where ineither case the array will have the characteristics described below.Arrays of nucleic acids are known in the art, where representativearrays that may be modified to become arrays of the subject invention asdescribed below, include those described in: U.S. Pat. Nos. 6,656,740;6,613,893; 6,599,693; 6,589,739; 6,587,579; 6,420,180; 6,387,636;6,309,875; 6,232,072; 6,221,653; and 6,180,351 and the references citedtherein.

The number of nucleic acid features of the initial or precursor arraymay vary, where the number of features present on the surface of thearray may be at least 2, 5, or 10 or more such as at least 20 andincluding at least 50, where the number may be as high as about 100, asabout 500, as about 1000, as about 5000, as about 10000 or higher. Inrepresentative embodiments, the subject arrays have a density rangingfrom about 100 to about 100,000 features/cm², such as from about 500 toabout 20,000 features/cm², including from about 1000 to about 20,000features/cm². In representative embodiments, the density ofsingle-stranded nucleic acids within a given feature is selected tooptimize efficiency of the RNA polymerase. In certain of theserepresentative embodiments, the density of the single-stranded nucleicacids may range from about 10⁻³ to about 1 pmol/mm², such as from about10⁻² to about 0.1 pmol/mm², including from about 5×10⁻² to about 0.1pmol/mm².

As mentioned above, each distinct surface immobilized nucleic acid ofthe features on the array includes a variable domain separated from thesurface of the solid support by a cleavable domain having a cleavablesite, e.g., residue, sequence, etc. The variable domains of the featuresof the precursor array have sequences that are chosen based on theparticular application in which the array is to be used, andspecifically the intended use of the product solution phase mixture offirst moieties produced using the array in accordance with the subjectmethods. The length of the variable domain may vary considerably andwill be chosen based on the intended use of the solution phase firstmoieties, and in representative embodiments, ranges from about 10 toabout 150 nt, such as from about 15 to about 100 nt and including fromabout 20 to about 80 nt.

As mentioned above, in addition to the variable domain, each surfaceimmobilized nucleic acid present on the array includes a cleavabledomain having a cleavable region. The cleavable region of the cleavabledomain may be cleavable by a number of different mechanisms. In certainembodiments, the cleavable domain includes a sequence of residues that,when present in duplex format, is recognized and cleaved by arestriction endonuclease. A large number of restriction endonucleasesare known to those of skill in the art. Specific restrictionendonuclease recognized sites of interest that may make up the subjectrecognition sequence include, but are not limited to: Hinc II and thelike. The length of the endonuclease recognition domain in theseembodiments may range from about 4 nucleotides to about 8 nucleotides,such as from about 5 nucleotides to about 6 nucleotides. In yet otherembodiments, the cleavable domain, and particularly the cleavable regionthereof, may be cleaved by light, i.e. photocleavable, or the domain maybe chemically cleavable, e.g., acid or base labile. In such domains, thedomain will comprise a cleavable moiety that is either photo orchemically cleavable. Photocleavable or photolabile moieties that may beincorporated into the constant domain may include, but are not limitedto: o-nitroarylmethine and arylaroylmethine, as well as derivativesthereof, and the like. Chemically cleavable moieties that may beincorporated into the constant domain may include, but are not limitedto: dialkoxysilane, β-cyano ether, amino carbamate, dithioacetal,disulfide, and the like.

In representative embodiments, each surface immobilized nucleic acid onthe array employed in the subject methods is described by the followingformula:surface-L-V-

wherein:

-   -   L is a cleavable domain having a cleavable region; and    -   V is said surface distal variable domain;    -   where each of the above features is as described above

In certain of these representative embodiments, only the variable domainV of said surface immobilized single-stranded nucleic acids differsbetween features. As indicated above, the single-stranded nucleic acidmay be oriented such that either the 3′ or 5′ end of the molecule isproximal to the substrate surface.

The subject arrays are provided by any convenient means, includingobtaining them from a commercial source or by synthesizing them de novo.To synthesize the arrays employed in the subject methods, the first stepis generally to determine the nature of the mixture of nucleic acidsthat is to be produced using the subject array according to the subjectmethods. For example, where the solution phase moieties are to beemployed as template directing the synthesis of product nucleic acids,the variable domains of the surface immobilized nucleic acids arechosen-based on the sequences of the desired product nucleic acids. Anyconvenient method may be employed to determine the sequences of thesurface immobilized nucleic acids, including probe design algorithms,including but not limited to those algorithms described in U.S. Pat. No.6,251,588 and published U.S. Application Nos. 20040101846; 20040101845;20040086880; 20040009484; 20040002070; 20030162183 and 20030054346; thedisclosures of which are herein incorporated by reference. Followingidentification of the probe sequences as defined above, an array isproduced in which each of the probe sequences of the identified ordesigned set of sequences is present.

Following provision of the array employed in the subject methods, asdescribed above, the next step is to cleave the surface immobilizednucleic acids of the array features from the solid support to produce asolution phase mixture of first moieties. In this step of the subjectmethods, the array is subjected to cleavage conditions sufficient tocleave the immobilized first nucleic acids of the features from thesubstrate surface. Generally, this step comprises contacting the arraywith an effective amount of a cleavage agent. The cleavage agent will,necessarily, be chosen in view of the particular nature of the cleavableregion of the cleavable domain that is to be cleaved, such that theregion is labile with respect to the chosen cleavage agent. Where thecleavable domain comprises a photocleavable or photolabile group,cleavage can be effectuated by subjecting the cleavable domain to lightof the appropriate wavelength sufficient to cleave the cleavable region.

Likewise, for chemically cleavable moieties, the array can be contactedwith a chemical capable of cleaving the linker, e.g. the appropriateacid or base, depending on the nature of the chemically labile moiety.Suitable cleavable sites include, but are not limited to, the following:base-cleavable sites such as esters, particularly succinates (cleavableby, for example, ammonia or trimethylamine), quaternary ammonium salts(cleavable by, for example, diisopropylamine) and urethanes (cleavableby aqueous sodium hydroxide); acid-cleavable sites such as benzylalcohol derivatives (cleavable using trifluoroacetic acid), teicoplaninaglycone (cleavable by trifluoroacetic acid followed by base), acetalsand thioacetals (also cleavable by trifluoroacetic acid), thioethers(cleavable, for example, by HF or cresol) and sulfonyls (cleavable bytrifluoromethane sulfonic acid, trifluoroacetic acid, thioanisole, orthe like); nucleophile-cleavable sites such as phthalamide (cleavable bysubstituted hydrazines), esters (cleavable by, for example, aluminumtrichloride); and Weinreb amide (cleavable by lithium aluminum hydride);and other types of chemically cleavable sites, includingphosphorothioate (cleavable by silver or mercuric ions) anddiisopropyldialkoxysilyl (cleavable by fluoride ions). Other cleavablesites will be apparent to those skilled in the art or are described inthe pertinent literature and texts (e.g., Brown (1997) ContemporaryOrganic Synthesis 4(3); 216-237).

Similarly, in those embodiments where the cleavable domain includes arestriction endonuclease recognized sequence, the array is contactedwith an effective amount of the appropriate restriction endonucleasethat recognizes and cleaves the sequence.

The above-described methods result in the production of a plurality ofsolution phase nucleic acids, where each of the different variabledomains of the precursor array is represented in the plurality, i.e.,for each feature present on the template array, there is at least onenucleic acid in the product plurality that corresponds to the feature,where by corresponds is meant that the nucleic acid is one that isgenerated by cleavage of a surface immobilized first nucleic acid of thefeature of the array. The length of each of the product nucleic acidspresent in the resultant plurality ranges, in representativeembodiments, from about 10 to about 1000 nt, such as from about 20 toabout 500 nt, including from about 30 to about 120 nt.

The plurality of nucleic acids produced in these embodiments of thesubject methods is characterized by having a known composition. By knowncomposition is meant that, because of the way in which the plurality isproduced, the sequence of each distinct nucleic acid in the productplurality can be predicted with a high degree of confidence.Accordingly, the sequence of each individual or distinct nucleic acid inthe product plurality is known. In many embodiments, the relative amountor copy number of each distinct nucleic acid of differing sequence inthe plurality is known. Put another way, the product plurality ofnucleic acids is known to include a constituent nucleic acidcorresponding to each feature of the precursor array used to produce it,such that each feature of the precursor array is represented in theproduct plurality.

In representative embodiments, the amount or copy number of eachdistinct nucleic acid of differing sequence in the product plurality isknown. The amounts of each distinct nucleic acid in the productplurality may be equimolar or non-equimolar, and are conveniently chosenand controlled by employing a precursor array with the desired number offeatures (as well as molecules per/feature) for each member of theplurality. For example, where a product plurality that is equimolar foreach member nucleic acid is desired, a precursor array with the samenumber of features for each member nucleic acid is employed.Alternatively, where a product plurality is desired in which there aretwice as many nucleic acids of a first sequence as compared to a secondsequence, a precursor array that has two times as many features of thefirst sequence as compared to the second sequence may be employed.

A feature of the nucleic acids of the product pluralities is that theyare single-stranded ribonucleic acids. As such, the nucleic acids of thesubject pluralities are not hybridized to complementary nucleic acids.In other words, the constituent nucleic acids of the product pluralitiesare not hybridized to separate nucleic acids of complementary sequence,where the separate nucleic acids are not covalently joined to them.While the product nucleic acids of the plurality are single-stranded,they may be linear or assume some secondary configuration, e.g., ahairpin configuration, and the like. The number of different or distinctnucleic acids present of differing sequence in the product plurality mayvary, but is generally at least 2, at least 5, at least 10, such as atleast about 20, at least about 50, at least about 100 or more, where thenumber may be as great as about 1000, about 5000 or about 25,000 orgreater. Any two given nucleic acids in the product pluralities areconsidered distinct or different if they include a stretch of at least20 nucleotides in length in which the sequence similarity is less then98%, as determined using the FASTA program (using default settings).

The product plurality of nucleic acids may be a heterogeneous mixture orset of individual homogeneous nucleic acid compositions, depending onthe intended use of the product plurality.

For those embodiments where the product plurality is a mixture, the termmixture refers to a heterogeneous composition of a plurality ofdifferent ribonucleic acids that differ from each other by residuesequence. Accordingly, the mixtures produced by the subject methods maybe viewed as compositions of two or more nucleic acids that are notchemically combined with each other and are capable of being separated,e.g., by using an array of complementary surface immobilized nucleicacids, but are not in fact separated.

In those embodiments where the plurality of nucleic acids is a set ofhomogenous nucleic acid populations, the constituent members of the setare, in certain embodiments, physically separated, such as present ondifferent locations of a solid support (e.g., of the precursor array),present in different containment structures, and the like.

The product pluralities of nucleic acids are physically separated fromthe precursor array as part of or following the cleavage step, asdescribed above. As such, the product of the first step of the subjectmethods is a solution phase mixture of nucleic acids.

The second step of the subject methods is to contact the solution phasemixture of nucleic acids with one or more reactants, as desired ornecessary, under conditions sufficient to produce the desired productmixture of second molecules, e.g., nucleic acids. In the broadest sense,the one or more reactants may be any single or combination of reactiveagents that, in the presence of the solution phase first moiety, e.g.,nucleic acid, result in the production of the desired product molecule,e.g., product nucleic acids.

In certain representative embodiments, the one or more reactantsemployed in this step include an enzymatic activity. While the enzymaticactivity may vary greatly, in representative embodiments the enzymaticactivity is a polymerase activity, particularly a template dependentpolymerase activity, such as an RNA or DNA polymerase. In theseembodiments, one or more reactants may make up an enzymatic reactionsystem, particularly a template dependent polymerase reaction system,where the system may or may not be an amplification system, where thesystem may include one or more additional reagents, such as NTPs, dNTPs,primers, buffers, mono- or divalent cations, etc., as illustrated ingreater detail below.

The particular conditions in which the solution phase first moieties arecontacted with the one or more reactants will, of course, vary,depending on the particular reaction to be performed. Illustrativeconditions are provided below in connection with a description ofrepresentative applications of the subject methods.

As mentioned earlier, the above provided discussion has been madeprimarily in terms of nucleic acid embodiments solely for ease ofdescription. As such, embodiments in which the array is an array ofother types of chemical moieties, such as polypeptides or otherbiopolymers, fall within the scope of the present invention, have beenenvisioned by the inventors at the time of filing the presentapplication (the present description of such being sufficient todemonstrate such) and are readily practiced by those of skill in the artbased on the above description.

The subject methods find use in a variety of different applications,representative applications of which are now reviewed in greater detailin the following section.

Utility

The subject methods of producing product second molecules using aprecursor array of surface immobilized first moieties find use in avariety of different applications, and are particularly suited for usein applications where a plurality of distinct or different biopolymers,such as nucleic acids or proteins, are to be simultaneously subjected orcontacted with the same one or more reactants to produce a product ofdiverse second molecules. In other words, the subject methods areparticularly suited for use is in applications where a commonreactant(s) is contacted at the same time with a plurality of distinctprecursor molecules to produce a plurality of distinct productmolecules. The representative applications described in greater detailbelow are conveniently characterized as nucleic acid or polypeptideapplications, depending on whether the precursor array is an array ofnucleic acids or an array of polypeptides.

Nucleic Acid Applications

In these representative applications, the precursor array that isemployed is an array of nucleic acids, as illustrated above. While avariety of different types of nucleic acid applications can be practicedby the subject methods, of interest are those applications described inU.S. application Ser. No. 09/628,472; the disclosure of which is hereinincorporated by reference, which applications are modified to employ thecomposition of cleaved, solution phase first nucleic acids produced bythe subject methods as a template composition, in lieu of the array ofsurface immobilized nucleic acids described therein. The applicationsdescribed in this publication are now reviewed in greater detail. Whenthese applications are performed according to the present invention, thearray surface immobilized precursor nucleic acids described therein arecleaved prior to production of the overhang comprising template nucleicacids, such that the template of the reaction is not in an “array”format but is in solution phase, as illustrated further below.

In these particular applications, following cleavage of the firstnucleic acids from the precursor array to produce a solution phasecomposition of first nucleic acids (referred to below as a templatecomposition), the resultant template composition is employed to generatemixtures of nucleic acids by a template dependent polymerase reaction,in which the solution phase composition of first nucleic acids isemployed as the template. The mixture of product nucleic acids producedby these applications is characterized by having a known composition. Assuch, at least the sequence of each individual or distinct nucleic acidsin the product mixture of differing sequences is known. Inrepresentative embodiments, the relative amount or copy number of eachdistinct nucleic acid of differing sequence in the product mixture isknown.

In these applications, each nucleic acid present in the mixture at leastincludes a variable domain that serves to distinguish it from any othernucleic acids in the mixture, i.e., any other nucleic acid that does nothave the identical sequence—any nucleic acid that is not its copy. Thevariable domain, S_(ij), is a nucleic acid that hybridizes understringent conditions to gene i at location j and is capable of servingas a primer in reverse transcription beginning at base j. The number ofdifferent variable domains, S_(ij), present in the mixture may vary, butis generally at least about 10, usually at least about 20 and moreusually at least about 50, where the number may be as great as 25,000 orgreater. In many embodiments, the number of different variable domainspresent in the mixture ranges from about 1000 to 25,000, usually fromabout 4,200 to 8,400. In addition to the distinguishing variable domain,the constituent members of the mixture may all share one or more domainsof common sequence, depending on the particular protocol employed togenerate the mixture, as described in greater detail below.

In the subject methods, the first step is generally to provide thesolution phase mixture of template nucleic acids, using the methods asdescribed above. Each distinct nucleic acid of the mixture of solutionphase first nucleic acids includes a constant domain and a complementvariable domain. The complement variable domain of each distinct probehas a sequence that is the complement of a variable or distinguishingdomain found in a constituent member of the mixture of nucleic acidsthat is produced by the subject methods as described above, where bycomplement is meant that the variable and complement variable sequenceshybridize under stringent conditions, e.g., as described above.

Because of the nature of the subject methods, as described below, eachdistinct complement variable domain will be represented in the nucleicacid mixture produced using the solution of first nucleic acids astemplate, i.e., the complement of each distinct complement variabledomain sequence will be found in the mixture of nucleic acids producedby the subject methods. For example, where a solution phase mixture offirst nucleic acids, i.e., a template composition, has 10 differentnucleic acids that differ by complement variable domain such that it has10 different complement variable domains, i.e., cV₁₋₁₀, the nucleic acidmixture produced by the subject methods as described below will have 10different or distinct nucleic acids, where each different nucleic acidsequence in the mixture includes a sequence that is the complement ofone of

-   -   cV₁₋₁₀, i.e., V_(1-10.)

As mentioned above, in addition to the unique complement variabledomain, each nucleic acid present in the template composition includes acommon or shared constant domain that is one side, e.g., 3′, of thecomplement variable domain. This constant domain may range in lengthfrom about 20 to about 50, such as from about 20 to about 45 andincluding from about 25 to about 40 nt. The constant domain typicallycomprises at least one of the following constant sub-domains: afunctional domain; a recognition domain and a linker domain. Inrepresentative embodiments, each solution phase nucleic acid contains atleast a recognition sub-domain, and optionally a functional domainand/or a linker domain. These constant sub-domains may be groupedtogether on the nucleic acid or separated so as to flank the variabledomain of the probe. As such, in certain embodiments these sub-domainsare generally arranged in the order of functional domain, recognitiondomain and linker domain going from the 5′ to the 3′ end of the nucleicacid sequence, such that the linker domain is at the 3′ terminus. In yetother embodiments, one or more of the domains, e.g., the functionalsub-domain, may be present on the 5′ end of the variable domain.

The optional functional sub-domain is generally a sequence that impartsor contributes some function to a duplex nucleic acid in which it ispresent. Functional domains of interest include: polymerase promotersites, e.g., T3 or T7 RNA polymerase promoter sites, sequences uniquewith respect to the intended target organism for the array experiment(i.e. unique priming sites) and the like. The length of this functionaldomain typically ranges from about 10 nt to about 40 nt, usually fromabout 20 nt to about 30 nt

The recognition sequence of the constant domain is typically a sequencethat, when present in duplex format, is recognized and cleaved by arestriction endonuclease. A large number of restriction endonucleasesare known to those of skill in the art. Specific restrictionendonuclease recognized sites of interest that may make up the subjectrecognition sequence include, but are not limited to: Hinc II and thelike. Generally, the length of the recognition domain ranges from about4 nt to about 8 nt, usually from about 5 nt to about 6 nt

The linker sub-domain of the subject constant domains is optional. Thelinker domain may be any convenient sequence, including random sequenceor a non-polynucleotide chemical linker (e.g. an ethylene glycol-basedpolyether oligomer), where the sole purpose of the linker domain is toproject the other domains of the probe away from the substrate surface.Generally, the linker domain if present, has a length ranging from about1 to about 20, including from about 1 to about 15, such as from about 1to about 10, for example from about 5 to about 10 nt.

In representative embodiments, each nucleic acid of the template mixtureemployed in the subject methods is described by the following formula:3′-X-R-F-cV-5′

wherein:

-   -   X is the optional linking domain;    -   R is the recognition domain;    -   F is the functional domain; and    -   cV is the complement variable domain, i.e., the complement of        the variable domain, cS_(ij), of the nucleic acid produced by        the subject methods to which it hybridizes under stringent        conditions;

where each of these elements are as described above.

Following provision of the template composition by the subject methods,as described above, the next step is to contact the solution phasetemplate composition with a universal primer under hybridizationconditions sufficient to produce a template composition that includes aplurality of overhang comprising duplex nucleic acids, where theoverhang is made up of the complement variable domain of each nucleicacid of the template composition. The universal primer is capable ofhybridizing to the constant domain, or at least a portion thereof (e.g.,at least that portion immediately 3′ of the complement variable domain).The universal primer has a length that is sufficient to prime templatedriven primer extension, where the length of the universal primergenerally ranges from about 10 to about 45 nt, such as from about 15 toabout 35 nt and including from about 20 to about 30 nt. Inrepresentative embodiments, the universal primer is the complement ofthe recognition and/or functional sub-domains of the constant domain ofeach nucleic acid in the template composition. As such, inrepresentative embodiments the universal primer that is employed has asequence described by the formula:5′-cR-cF-3′

wherein:

-   -   cR is the complement of the recognition domain; and    -   cF is the complement of the functional domain.

As mentioned above, the template composition following contact with theuniversal primer, as described above, is made up of duplex moleculesmade up of a first nucleic acid having a constant and complementvariable domain and a second nucleic acid which is the universal primerand is hybridized to the constant domain (or at least that portion ofthe constant domain that is 3′ of the variable domain complement). Assuch, the solution phase composition produced by this step is acomposition of overhang comprising duplex nucleic acids, typically DNA,molecules, where the overhang is made up of the complement variabledomain of each nucleic acid in the template composition.

This composition of overhang comprising duplex nucleic acids is thensubjected to template dependent polymerase mediated reaction conditionssufficient to produce the desired mixture of product nucleic acids. Thespecific reaction conditions to which the composition of overhangcomprising duplex nucleic acids is subjected may vary depending on theparticular protocol used and/or the specific nature of the nucleic acidmixture to be produced therefrom. Specific reaction conditions ofinterest include, but are not limited to: linear PCR (Polymerase ChainReaction); strand displacement amplification; and in vitrotranscription. Each of these specific reaction conditions is nowreviewed in greater detail.

Where the overhang comprising duplex nucleic acid composition issubjected to linear PCR conditions, the composition is contacted in anaqueous reaction mixture with a source of DNA polymerase, dNTPs and anyother desired or requisite reagents under conditions sufficient toproduce linearly amplified amounts of nucleic acids, e.g., under thermalcycling conditions. As such, the polymerase employed in the subjectmethods is generally, though not necessarily (e.g., where new polymeraseis added after each cycle) a thermostable polymerase. A variety ofthermostable polymerases are known to those of skill in the art, whererepresentative polymerases include, but are not limited to: Taqpolymerase, Vent® polymerase, Pfu polymerase and the like. The amount ofpolymerase present in the reaction mixture may vary but is sufficient toprovide for the requisite amount of polymerase activity, where thespecific amount employed may be readily determined by those of skill inthe art. Also present in the reaction mixture is a collection of thefour dNTPs, i.e., dATP, dCTP, dGTP and dTTP. The dNTPs may be present invarying or equimolar amounts, where the amount of each dNTP typicallyranges from about 10 μM to 10 mM, usually from about 100 μM to 300 μM.Other reagents that may be present in the reaction mixture include:monovalent cations (e.g. Na⁺), divalent cations (e.g. Mg⁺⁺), buffers(e.g. Tris), surfactants (e.g. Triton X-100) and the like. In thislinear PCR embodiment of the subject methods, the reaction mixture issubjected to thermal cycling conditions in which the temperature of thereaction mixture is cycled through an annealing, primer extension anddissociation temperatures in a manner that results in the production oflinearly amplified amounts of nucleic acid for each different sequenceprobe on the template composition. In representative embodiments, theannealing temperature typically ranges from about 50° C. to 80° C.,usually from about 60° C. to 75° C. and is maintained for period of timeranging from about 10 sec. to 10 min., usually from about 30 sec. to 2min. The primer extension temperature typically ranges from about 55° C.to 75° C., usually from about 60° C. to 70° C. and is maintained forperiod of time ranging from about 30 sec. to 10 min., usually from about1 min. to 5 min. The dissociation temperature typically ranges fromabout 80° C. to 99° C., usually from about 90° C. to 95° C. and ismaintained for period of time ranging from about 1 sec. to 2 min.,usually from about 30 sec. to 1 min.

In strand displacement amplification, the composition of overhangcomprising duplex nucleic acids is employed as primed template in linearamplification variations of the exponential amplification protocolsdescribed in Walker et al., Nucleic Acids Res. (1992) 20:1691-1696 andWalker et al., Proc. Nat'l Acad. Sci. USA (1992) 89:392-396; as well asin U.S. Pat. No. 5,648,211; the disclosure of which is hereinincorporated by reference. Briefly, isothermal linear amplification isachieved as follows. Following production of the composition of overhangcomprising duplex nucleic acids, the composition is subjected to a cycleof strand nicking of the universal primer after sequence cR, typicallyby using a restriction endonuclease. Generally, the template strand orprobe sequence is protected via an appropriately placed phosphorthioatelinkage in the surface-bound template strand. Extension of the 3′ endexposed by the nick is then allowed to proceed by using a DNA polymerasethat lacks a 5′→3′ exonuclease activity but possesses a stranddisplacement activity, e.g., Klenow fragment. Each cycle in thisprotocol releases a nucleic acid molecule which has the formula:5′-cF-Sij-3′. (It is noted that, depending on the nature of the initialarray, the polarity of the product molecule may be reversed, e.g., themolecule may have the formula 3′-cF-Sij-5′.) In certain variants of thismethod, nicking may be achieved by making R a half-site for arestriction endonuclease that exhibits single-strand cleavage activity,or by employing a nicking endonuclease, such as N.BstNBI, and the like.

In yet other embodiments, the subject composition of overhang comprisingduplex nucleic acids is employed in an in vitro transcription method. Inthis embodiment, the template composition may be one that includesnucleic acid molecules of the following formula:3′-X-R-(C)Sij-F-5′

wherein:

-   -   X and R are as defined above;    -   F is an RNA polymerase promoter, e.g., T3 or T7 promoter; and    -   (C) Sij is Sij modified to end in a C residue.

The universal primer employed with this composition has the formula5′-cR-3′. When the template composition is contacted with NTPs, T3 or T7polymerase and the appropriate transcription buffer, ribonucleic acidsof the formula: 5′-(rG)rcSij-rcF-3′ are produced, where r stands forribonucleotide. (It is noted that, depending on the nature of theinitial array, the polarity of the product molecule may be reversed,e.g., the molecule may have the formula 3′-(rG)rcSij-rcF-5′.) Bycontacting this resultant mixture of ribonucleic acids with the DNAprimer 5′-F-3′ and a reverse transcriptase, a mixture ofdeoxyribonucleic acids suitable for use as primer in target generationprotocols is produced.

The above described representative methods result in the production of amixture of nucleic acids, typically a mixture of deoxyribonucleic acids,where each of the different complement variable domains of the templatecomposition is represented in the mixture, i.e., there is at least onenucleic acid in the mixture that has a variable domain that hybridizesunder stringent conditions to each different complement variable domainpresent on the template composition.

The nucleic acid mixtures produced in these representative applicationsfind use in a variety of different applications, and are particularlysuited for use as primers in the generation of target nucleic acids,e.g., for array based differential gene expression analysisapplications. Where the subject nucleic acids mixtures are used asprimers for target generation in gene expression analyses, the firststep is to generate a population of target nucleic acids from an initialmRNA source or sample. By target nucleic acid is meant a nucleic acidthat has a sequence, e.g., S_(ij), which is either the same as, orcomplementary to, the sequence of an mRNA found in an initial sample,where the target may be DNA or RNA and be present in amplified amountsas compared to the initial amount of mRNA, depending on the particulartarget generation protocol that is employed.

In these embodiments, the target or image nucleic acids are producedfrom the subject nucleic acid mixtures generally through enzymaticgeneration protocols. Specifically, the target nucleic acids aretypically produced using template dependent polymerization protocols andan initial mRNA source. The initial mRNA source may be present in avariety of different samples, where the sample will typically be derivedfrom a physiological source. The physiological source may be derivedfrom a variety of eukaryotic or prokaryotic sources, with physiologicalsources of interest including sources derived from single-celledorganisms such as yeast and multicellular organisms, including plantsand animals, particularly mammals, where the physiological sources frommulticellular organisms may be derived from particular organs or tissuesof the multicellular organism, or from isolated cells derived therefrom.In obtaining the sample of RNA to be analyzed from the physiologicalsource from which it is derived, the physiological source may besubjected to a number of different processing steps, where suchprocessing steps might include tissue homogenization, cell isolation andcytoplasm extraction, nucleic acid extraction and the like, where suchprocessing steps are known to those of skill in the art. Methods ofisolating RNA from cells, tissues, organs or whole organisms are knownto those of skill in the art and are described in Maniatis et al.(1989), Molecular Cloning: A Laboratory Manual 2d Ed. (Cold SpringHarbor Press).

A number of different enzymatic protocols for generating image or targetnucleic acids from an initial mRNA sample are known and continue to bedeveloped. Any convenient protocol may be employed, where the particularprotocol employed depends, at least in part, on a number of factors,including: whether one wants to generate amplified amounts of target orimage nucleic acid; whether one wants to generate geometrically orlinearly amplified amounts of target nucleic acid; whether bias in theamount of target can be tolerated, etc. A common feature of theserepresentative applications is the use of the subject nucleic acidmixtures produced using the above described protocols as primer.

A number of nucleic acid amplification methods can be employed togenerate the target nucleic acid from an initial mRNA source, wherethese methods can employ the subject nucleic acid mixtures as primer.Such methods include the “polymerase chain reaction” (PCR) as describedin U.S. Pat. No. 4,683,195, the disclosure of which is hereinincorporated by reference, and a number of transcription-basedexponential amplification methods, such as those described in U.S. Pat.Nos. 5,130,238; 5,399,491; and 5,437,990; the disclosures of which areherein incorporated by reference. Each of these methods usesprimer-dependent nucleic acid synthesis to generate a DNA or RNAproduct, which serves as a template for subsequent rounds ofprimer-dependent nucleic acid synthesis. Each process uses (at least)two primer sequences complementary to different strands of a desirednucleic acid sequence and results in an exponential increase in thenumber of copies of the target sequence.

Alternatively, amplification methods that utilize a single primer may beemployed to generate target or image nucleic acids from an initial mRNAsample, where the subject nucleic acid mixtures are employed as primer.See e.g. U.S. Pat. Nos. 5,554,516; and 5,716,785; the disclosures ofwhich are herein incorporated by reference. The methods reported inthese patents utilize a single primer containing an RNA polymerasepromoter sequence and a sequence complementary to the 3′-end of thedesired nucleic acid target sequence(s) (“promoter-primer”). In bothmethods, the promoter-primer is added under conditions where ithybridizes to the target sequence(s) and is converted to a substrate forRNA polymerase. In both methods, the substrate intermediate isrecognized by RNA polymerase, which produces multiple copies of RNAcomplementary to the target sequence(s) (“cRNA”).

Whatever process is employed to generate the target nucleic acid, whererepresentative protocols have been provided immediately above, theprocess may be modified to include the use of chemical analogs ofnucleotides that have been modified to include a label moiety, e.g., anorganic fluorophore, an isotopic label, a capture ligand, e.g., biotin,etc. As a result, the target nucleic acids produced using the subjectnucleic acid mixtures as primers often are labeled, either directly orindirectly, for use in subsequent hybridization assays.

The above target generation protocols are merely representative and byno means inclusive of all of the different types of protocols in whichthe subject nucleic acid mixtures find use as primers.

The resultant populations of target nucleic acids find use as, interalia, target in hybridization assays, such as gene expression analysisapplications. Gene expression analysis protocols are well known to thoseof skill in the art, and the populations of target nucleic acidsproduced by the subject methods find use in many, if not all, of theseprotocols. In gene expression analysis protocols using the subjectpopulations of labeled target, the population of labeled target istypically contacted with a population of probe nucleic acids, e.g., onan array, under hybridization conditions, usually stringenthybridization conditions. Following hybridization, non-bound target isremoved or separated from the probe, e.g., by washing. Washing resultsin a pattern of hybridized target, which may be read using anyconvenient protocol, e.g., with a fluorescent scanner device. From thispattern, information regarding the mRNA expression profile in theinitial mRNA sample from which the target population was produced may bereadily derived or deduced.

In certain embodiments, the subject methods include a step oftransmitting data from at least one of the detecting and deriving steps,as described above, to a remote location. By “remote location” is meanta location other than the location at which the array is present andhybridization occur. For example, a remote location could be anotherlocation (e.g. office, lab, etc.) in the same city, another location ina different city, another location in a different state, anotherlocation in a different country, etc. The data may be transmitted to theremote location for further evaluation and/or use. Any convenienttelecommunications means may be employed for transmitting the data,e.g., facsimile, modem, internet, etc.

In addition to the above described representative applications in whichtemplate compositions of solution phase nucleic acids produced by thesubject methods are used to generate mixtures of deoxyribonucleic acids,the methods of the present invention may also be used to generatetemplate compositions of nucleic acids that are subsequently employed togenerate pluralities of ribonucleic acids, as described in copendingapplication Ser. No. 10/961,991 filed on Oct. 8, 2004.

In these representative applications, the subject methods are employedto produce a template composition of first nucleic acids, where eachnucleic acid includes a RNA polymerase promoter domain and a variabledomain. The variable domains of the features of the precursor array havesequences that are chosen based on the particular application in whichthe array is to be used, and specifically the intended use of theribonucleic acid mixture produced using the array in accordance with thesubject methods. The length of the variable domain may vary considerablyand will be chosen based on the desired length of the resultantribonucleic acids in the to be produced RNA composition within thesynthesis constraints of the subject method. In representativeembodiments, the length of the variable domain will range from about 10to about 150 nt, such as from about 15 to about 100 nt and includingfrom about 20 to about 80 nt.

Each of the nucleic acids of the template composition also includes anRNA polymerase promoter domain, which domain may be the same ordifferent between or among the constituent members of the templatecomposition. Suitable promoter domains or regions that find use in thesubject methods are ones that are capable of initiating transcription ofan operationally linked DNA sequence in the presence of ribonucleotidesand an RNA polymerase under suitable conditions. The promoter domain orregion is linked in an orientation to permit transcription of RNA, asdescribed in greater detail below. The promoter region may includebetween about 15 and about 250 nucleotides, such as between about 17 andabout 60 nucleotides, from a naturally occurring RNA polymerase promoteror a consensus promoter region, as described in Alberts et al. (1989) inMolecular Biology of the Cell, 2d Ed. (Garland Publishing, Inc.).Prokaryotic promoters or eukaryotic promoters may be employed, and inrepresentative embodiments prokaryotic promoters are employed, such asphage or virus promoters. As used herein, the term “operably linked”refers to a functional linkage between the affecting sequence (typicallya promoter) and the controlled sequence, e.g., the variable domain. Thepromoter regions that find use are regions where RNA polymerase bindstightly to the DNA and contain the start site and signal for RNAsynthesis to begin. A wide variety of promoters are known and many arevery well characterized. Representative promoter regions of interestinclude, but are not limited to: T7, T3 and SP6 as described inChamberlin and Ryan, The Enzymes (ed. P. Boyer, Academic Press, NewYork) (1982) pp 87-108.

In these representative applications, the above-described templatecomposition is then contacted with a RNA polymerase promoter complementcomposition under conditions sufficient to produce a composition ofoverhang containing duplex nucleic acids. The RNA polymerase promotercomplement composition is a nucleic acid composition that is made up ofone or more distinct types nucleic acids of different sequence, where agiven nucleic acid member of the complement composition is capable ofhybridizing to an RNA polymerase promoter domain present on a nucleicacid in the template composition. The complement composition may behomogeneous or heterogeneous, depending on whether there is a single RNApolymerase promoter domain represented in the template composition, or aplurality of different such promoter domains. The nucleic acid membersof the complement composition have a length that is sufficient to bindto the complementary domain of the nucleic acids present in the templatecomposition and produce a functional RNA polymerase promoter site, wherethe length of the constituent nucleic acid members may range from about10 to about 45 nt, such as from about 15 to about 35 nt and includingfrom about 20 to about 30 nt.

As mentioned above, the composition produced by this method iscomposition of duplex probe molecules made up of a first nucleic acidhaving a constant and complement variable domain and a second nucleicacid which is the universal primer and is hybridized to the constantdomain (or at least that portion of the constant domain that is 3′ ofthe variable domain complement). As such, the composition produced bythis step is a composition of overhang comprising duplex nucleic acid,typically DNA, molecules, where the overhang is made up of thecomplement variable domain of each nucleic acid in the composition.

Optionally, the resultant composition of overhang comprising duplexsurface immobilized nucleic acids or probes is then subjected to primerextension reaction conditions sufficient to produce a composition offull-length duplex nucleic acids. The specific primer extension reactionconditions to which the composition of overhang comprising duplexnucleic acids is subjected may vary, so long as the conditions producethe desired surface immobilized duplex nucleic acids. In representativeembodiments, the composition is contacted in an aqueous reaction mixturewith a source of DNA polymerase, dNTPs and any other desired orrequisite primer extension reagents under conditions sufficient toproduce the desired surface immobilized duplex nucleic acids.

Following production of the template composition, e.g., overhang oroptional duplex template compositions, as described above, the resultanttemplate composition is then subjected to in vitro transcriptionreaction conditions sufficient to produce the desired productribonucleic acid plurality. During this step, the at least partiallyduplex DNAs produced in the first step of the methods are transcribed byRNA polymerase to yield RNA product. In this step, the at leastpartially duplex DNAs are contacted with the appropriate RNA polymerasein the presence of the four ribonucleotides, under conditions sufficientfor RNA transcription to occur, where the particular polymerase employedwill be chosen based on the promoter region present in thedouble-stranded DNA, e.g. T7 RNA polymerase, T3 or SP6 RNA polymerases,E. coli RNA polymerase, and the like. Suitable conditions for RNAtranscription using RNA polymerases are known in the art, see e.g.Milligan and Uhlenbeck (1989), Methods in Enzymol. 180, 51.

Where desired, the RNA pluralities that are produced by the subjectmethods may be produced as labeled pluralities of RNAs. The label may beincorporated into the product RNAs using any convenient protocol, e.g.,by employing labeled NTPs in the in vitro transcription reactionmixture, or by employing labeled RNA polymerase promoter complements.

These representative applications produce a plurality of ribonucleicacids, where each of the different variable domains of the templatecomposition is represented in the plurality, i.e., for each nucleic acidpresent in the template composition, there is at least one ribonucleicacid in the plurality that corresponds to the feature, where bycorresponds is meant that the nucleic acid is one that is generated byin vitro transcription using the variable domain of the feature astemplate. The length of each of the product ribonucleic acids present inthe resultant plurality ranges, in representative embodiments, fromabout 10 to about 10000 nt, such as from about 100 to about 6000 nt,including from about 300 to about 2000 nt.

The product RNA pluralities of these representative applications finduse in a variety of different applications, including, but not limitedto: gene expression applications, e.g., as reference or controlcompositions, or in the estimation and/or correction of background; andgene-silencing applications, e.g., as RNAi agents. These applicationsare further described in copending application Ser. No. 10/961,991 filedon Oct. 8, 2004.

Polypeptide Applications

The subject methods of producing a population of product molecules froma precursor array of surface immobilized first moieties also find use inpolypeptide applications. For example, an initial or precursor arrayhaving a plurality of distinct or different polypeptides immobilized ona surface thereof may be subjected to cleavage conditions to produce aplurality of solution phase polypeptides, which composition may then becontacted with a conjugation reaction system that chemically bonds acommon moiety of interest to each of the members of the solution phasecomposition to produce a plurality of second molecules, e.g.,polypeptide conjugates. Polypeptide precursor arrays of interestinclude, but are not limited to, arrays in which the polypeptide agentsare chosen from affibodies, antigens, antigen-binding sites, antibodies,as well as binding fragments and mimetics thereof (e.g., fAB, scFV,etc.), receptors, ligands, protease targets, targets for kinases, etc.The polypeptide moieties may bind to a variety of different targets,including, but not limited to: affibodies, antigens, antigen-bindingsites, antibodies, as well as binding fragments and mimetics thereof(e.g., fAB, scFV, etc.), receptors, ligands, protease targets, targetsfor kinases, as well as nucleic acid molecules, e.g., DNA, RNA includingsiRNAs, miRNAs, snRNAs, etc, aptamers, etc., and the like. In certainembodiments, the methods may further include a step of removing unboundpolypeptides from the solution phase, following which the boundmolecules may be detected by spectrally distinguishable labels,identifier tags included on a bound complex (which could be a label orbiopolymer acting as a bar code for individual molecules), byimmunoprecipitation, etc.

It is noted that the above reviewed nucleic acid and polypeptideapplications are merely representative of the diverse types ofapplications in which the subject methods find use, and that the subjectmethods are not limited to use merely in the above representativeapplications.

Kits

Also provided by the subject invention are kits for use in practicingthe subject methods. Generally, the kits include a precursor array, asdescribed above, and a cleavage reagent. Depending on the particularapplication in which the kits are to be employed, the kits may furtherinclude additional containers, each with one or more of the variousreagents (typically in concentrated form) utilized in specificapplications, including, for example, buffers, dNTPs, polymerase,labeled dNTPs, etc.

A set of instructions will also typically be included, where theinstructions may be associated with a package insert and/or thepackaging of the kit or the components thereof. These instructions maybe present in the subject kits in a variety of forms, one or more ofwhich may be present in the kit. One form in which these instructionsmay be present is as printed information on a suitable medium orsubstrate, e.g., a piece or pieces of paper on which the information isprinted, in the packaging of the kit, in a package insert, etc. Yetanother means would be a computer readable medium, e.g., diskette, CD,etc., on which the information has been recorded. Yet another means thatmay be present is a website address which may be used via the internetto access the information at a removed site. Any convenient means may bepresent in the kits.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the scope ofthe appended claims.

1. A method comprising: (a) subjecting an array of a plurality ofpolypeptide or polysaccharide features comprising polypeptide orpolysaccharide first moieties immobilized on a surface of a solidsupport via a cleavable domain to conditions sufficient to cleave saidcleavable domain and generate a solution phase composition of saidpolypeptide or polysaccharide first moieties, wherein said solutionphase composition is a mixture comprising two or more of saidpolypeptide or polysaccharide first moieties; and (b) contacting saidsolution phase composition of said polypeptide or polysaccharide firstmoieties with one or more reactants to produce a mixture of productmolecules that are different from said polypeptide or polysaccharidefirst moieties.
 2. The method according to claim 1, wherein said arrayis an array of a plurality of polysaccharide features.
 3. The methodaccording to claim 1, wherein said cleavable domain is base labiledomain.
 4. The method according to claim 1, wherein said cleavablelinker is an acid labile linker.
 5. The method according to claim 1,wherein said one or more reactants comprises an enzymatic activity. 6.The method according to claim 5, wherein said product molecules arepolypeptides.
 7. A method comprising: (a) subjecting an array of aplurality of polypeptide features each comprising first polypeptidesimmobilized on a surface of a solid support via a cleavable domain toconditions sufficient to cleave said cleavable domain and generate asolution phase composition of said first polypeptides, wherein saidsolution phase composition is a mixture comprising two or more of saidfirst polypeptides; and (b) contacting said solution phase compositionof said first polypeptides with an enzymatic reagent system to produce amixture of product polypeptides that are different from said firstpolypeptides.
 8. The method according to claim 7, wherein said cleavabledomain comprises base labile region.
 9. The method according to claim 7,wherein said cleavable domain comprises an acid labile region.